Barabino S M, Sproat B S, Ryder U, Blencowe B J, Lamond A I
European Molecular Biology Laboratory, Heidelberg, FRG.
EMBO J. 1989 Dec 20;8(13):4171-8. doi: 10.1002/j.1460-2075.1989.tb08602.x.
Biotinylated 2'-OMe RNA oligonucleotides complementary to two separate regions of human U2 snRNA have been used as affinity probes to study U2 snRNP--pre-mRNA interactions. Both oligonucleotides bind specifically and allow highly selective removal of U2 snRNP from HeLa cell nuclear extracts. Pre-mRNA substrates can also be specifically affinity selected through oligonucleotides binding to U2 snRNP particles in splicing complexes. Stable binding of U2 snRNP to pre-mRNA is blocked by the pre-binding of an oligonucleotide to the branch site complementary region of U2 snRNA, but not by an oligonucleotide binding to the 5' terminus of U2. Both oligonucleotides affinity select the intron product, but not the intron intermediate, when added after spliceosome assembly has taken place. The effect of 2'-OMe RNA oligonucleotides on splicing complex formation has been used to demonstrate that complexes containing U2 snRNP and unspliced pre-mRNA are precursors to functional spliceosomes.
与人类U2 snRNA两个不同区域互补的生物素化2'-O-甲基RNA寡核苷酸已被用作亲和探针，以研究U2 snRNP与前体mRNA的相互作用。两种寡核苷酸都能特异性结合，并能从HeLa细胞核提取物中高度选择性地去除U2 snRNP。前体mRNA底物也可以通过与剪接复合物中U2 snRNP颗粒结合的寡核苷酸进行特异性亲和选择。U2 snRNP与前体mRNA的稳定结合被寡核苷酸与U2 snRNA分支位点互补区域的预结合所阻断，但不被与U2 5'末端结合的寡核苷酸所阻断。当剪接体组装完成后添加时，两种寡核苷酸都能亲和选择内含子产物，而不是内含子中间体。2'-O-甲基RNA寡核苷酸对剪接复合物形成的影响已被用于证明含有U2 snRNP和未剪接前体mRNA的复合物是功能性剪接体的前体。
